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Stephen G Waxman - One of the best experts on this subject based on the ideXlab platform.

  • a 49 residue sequence motif in the c terminus of nav1 9 regulates trafficking of the channel to the plasma membrane
    Journal of Biological Chemistry, 2020
    Co-Authors: Daria V Sizova, Mark Estacion, Stephen G Waxman, Jianying Huang, Elizabeth J Akin, Carolina Gomisperez, Sulayman D Dibhajj
    Abstract:

    : Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems, for example in human embryonic kidney 293 (HEK293) cells, has hampered studies of its biophysical and pharmacological properties, and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C-terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C-terminus where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

  • sodium channel nav1 9 mutations associated with insensitivity to pain dampen neuronal excitability
    Journal of Clinical Investigation, 2017
    Co-Authors: Jianying Huang, Stephen G Waxman, Carlos G Vanoye, Alison Cutts, Paul Y Goldberg, Sulayman D Dibhajj, Charles Jay Cohen, Alfred L. George
    Abstract:

    : Voltage-gated sodium channel (NaV) mutations cause genetic pain disorders that range from severe paroxysmal pain to a congenital inability to sense pain. Previous studies on NaV1.7 and NaV1.8 established clear relationships between perturbations in channel function and divergent clinical phenotypes. By contrast, studies of NaV1.9 mutations have not revealed a clear relationship of channel dysfunction with the associated and contrasting clinical phenotypes. Here, we have elucidated the functional consequences of a NaV1.9 mutation (L1302F) that is associated with insensitivity to pain. We investigated the effects of L1302F and a previously reported mutation (L811P) on neuronal excitability. In transfected heterologous cells, the L1302F mutation caused a large hyperpolarizing shift in the voltage-dependence of activation, leading to substantially enhanced overlap between activation and steady-state inactivation relationships. In transfected small rat dorsal root ganglion neurons, expression of L1302F and L811P evoked large depolarizations of the resting membrane potential and impaired action potential generation. Therefore, our findings implicate a cellular loss of function as the basis for impaired pain sensation. We further demonstrated that a U-shaped relationship between the resting potential and the neuronal action potential threshold explains why NaV1.9 mutations that evoke small degrees of membrane depolarization cause hyperexcitability and familial episodic pain disorder or painful neuropathy, while mutations evoking larger membrane depolarizations cause hypoexcitability and insensitivity to pain.

  • ca2 toxicity due to reverse na ca2 exchange contributes to degeneration of neurites of drg neurons induced by a neuropathy associated nav1 7 mutation
    Journal of Neurophysiology, 2015
    Co-Authors: Mark Estacion, Joel A. Black, Janneke G J Hoeijmakers, Catharina G Faber, Ingemar S J Merkies, Bhupinder P S Vohra, Guiseppe Lauria, Stephen G Waxman
    Abstract:

    Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na+ concentration ([Na+]) and intracellular [Ca2+] following stimulation with high [K+] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca2+] transients evoked by high [K+] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K+] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K+] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca2+ or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K+] and 2-DG. These results point to [Na+] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca2+ toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy.

  • Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus
    Experimental Neurology, 2014
    Co-Authors: Joel A. Black, Sulayman D. Dib-hajj, Dymtro Vasylyev, Stephen G Waxman
    Abstract:

    Abstract Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, NaV1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as “peripheral” sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies.

  • nav1 7 stress induced changes in immunoreactivity within magnocellular neurosecretory neurons of the supraoptic nucleus
    Molecular Pain, 2013
    Co-Authors: Joel A. Black, Janneke G J Hoeijmakers, Catharina G Faber, Ingemar S J Merkies, Stephen G Waxman
    Abstract:

    Background: NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a “peripheral” sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, Nav1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. Results: Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. Conclusions: NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.

Joel A. Black - One of the best experts on this subject based on the ideXlab platform.

  • ca2 toxicity due to reverse na ca2 exchange contributes to degeneration of neurites of drg neurons induced by a neuropathy associated nav1 7 mutation
    Journal of Neurophysiology, 2015
    Co-Authors: Mark Estacion, Joel A. Black, Janneke G J Hoeijmakers, Catharina G Faber, Ingemar S J Merkies, Bhupinder P S Vohra, Guiseppe Lauria, Stephen G Waxman
    Abstract:

    Gain-of-function missense mutations in voltage-gated sodium channel Nav1.7 have been linked to small-fiber neuropathy, which is characterized by burning pain, dysautonomia and a loss of intraepidermal nerve fibers. However, the mechanistic cascades linking Nav1.7 mutations to axonal degeneration are incompletely understood. The G856D mutation in Nav1.7 produces robust changes in channel biophysical properties, including hyperpolarized activation, depolarized inactivation, and enhanced ramp and persistent currents, which contribute to the hyperexcitability exhibited by neurons containing Nav1.8. We report here that cell bodies and neurites of dorsal root ganglion (DRG) neurons transfected with G856D display increased levels of intracellular Na+ concentration ([Na+]) and intracellular [Ca2+] following stimulation with high [K+] compared with wild-type (WT) Nav1.7-expressing neurons. Blockade of reverse mode of the sodium/calcium exchanger (NCX) or of sodium channels attenuates [Ca2+] transients evoked by high [K+] in G856D-expressing DRG cell bodies and neurites. We also show that treatment of WT or G856D-expressing neurites with high [K+] or 2-deoxyglucose (2-DG) does not elicit degeneration of these neurites, but that high [K+] and 2-DG in combination evokes degeneration of G856D neurites but not WT neurites. Our results also demonstrate that 0 Ca2+ or blockade of reverse mode of NCX protects G856D-expressing neurites from degeneration when exposed to high [K+] and 2-DG. These results point to [Na+] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca2+ toxicity, and suggest subtype-specific blockade of Nav1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy.

  • Nav1.9 expression in magnocellular neurosecretory cells of supraoptic nucleus
    Experimental Neurology, 2014
    Co-Authors: Joel A. Black, Sulayman D. Dib-hajj, Dymtro Vasylyev, Stephen G Waxman
    Abstract:

    Abstract Osmoregulation in mammals is tightly controlled by the release of vasopressin and oxytocin from magnocellular neurosecretory cells (MSC) of the supraoptic nucleus (SON). The release of vasopressin and oxytocin in the neurohypophysis by axons of MSC is regulated by bursting activity of these neurons, which is influenced by multiple sources, including intrinsic membrane properties, paracrine contributions of glial cells, and extrinsic synaptic inputs. Previous work has shown that bursting activity of MSC is tetrodotoxin (TTX)-sensitive, and that TTX-S sodium channels Nav1.2, Nav1.6 and Nav1.7 are expressed by MSC and upregulated in response to osmotic challenge in rats. The TTX-resistant sodium channels, NaV1.8 and Nav1.9, are preferentially expressed, at relatively high levels, in peripheral neurons, where their properties are linked to repetitive firing and subthreshold electrogenesis, respectively, and are often referred to as “peripheral” sodium channels. Both sodium channels have been implicated in pain pathways, and are under study as potential therapeutic targets for pain medications which might be expected to have minimal CNS side effects. We show here, however, that Nav1.9 is expressed by vasopressin- and oxytocin-producing MSC of the rat supraoptic nucleus (SON). We also show that cultured MSC exhibit sodium currents that have characteristics of Nav1.9 channels. In contrast, Nav1.8 is not detectable in the SON. These results suggest that Nav1.9 may contribute to the firing pattern of MSC of the SON, and that careful assessment of hypothalamic function be performed as NaV1.9 blocking agents are studied as potential pain therapies.

  • nav1 7 stress induced changes in immunoreactivity within magnocellular neurosecretory neurons of the supraoptic nucleus
    Molecular Pain, 2013
    Co-Authors: Joel A. Black, Janneke G J Hoeijmakers, Catharina G Faber, Ingemar S J Merkies, Stephen G Waxman
    Abstract:

    Background: NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a “peripheral” sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, Nav1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. Results: Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. Conclusions: NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.

  • Sodium Channel Expression Within Chronic Multiple Sclerosis Plaques
    Journal of Neuropathology and Experimental Neurology, 2007
    Co-Authors: Joel A. Black, Jia Newcombe, Bruce D. Trapp, Stephen G Waxman
    Abstract:

    Multiple sclerosis (MS) is characterized by focal destruction of myelin sheaths, gliotic scars, and axonal damage that contributes to the accumulation of nonremitting clinical deficits. Previous studies have demonstrated coexpression of sodium channel Nav1.6 and the sodium-calcium exchanger (NCX), together with β-amyloid precursor protein (β-APP), a marker of axonal damage, in degenerating axons within acute MS lesions. Axonal degeneration is less frequent within chronic MS lesions than in acute plaques, although current evidence suggests that axonal loss in chronic lesions ("slow burn") is a major contributor to accumulating disability. It is not known, however, whether axonal degenerations in chronic and acute lesions share common mechanisms, despite radically differing extracellular milieus. In this study, the expression of sodium channels Nav1.2 and Nav1.6 and of NCX was examined in chronic MS plaques within the spinal cord. Nav1.2 immunostaining was not observed along demyelinated axons in chronic lesions but was expressed by scar and reactive astrocytes within the plaque. Nav1.6 immunoreactivity, which was intense at nodes of Ranvier in normal appearing white matter in the same sections, was present in approximately one-third of the demyelinated axons within these plaques in a patchy rather than continuous distribution. NCX was not detected in demyelinated axons within chronic lesions, although it was clearly present within the scar astrocytes surrounding the demyelinated axons. β-APP accumulation occurred in a small percentage of axons within chronic lesions within the spinal cord, but β-APP was not preferentially present in axons that expressed Nav1.6. These observations suggest that different mechanisms underlie axonal degeneration in acute and chronic MS lesions, with axonal injury occurring at sites of coexpression of Nav1.6 and NCX in acute lesions but independent of coexpression of these 2 molecules in chronic lesions.

  • Contactin Associates with Sodium Channel Nav1.3 in Native Tissues and Increases Channel Density at the Cell Surface
    The Journal of Neuroscience, 2004
    Co-Authors: Bhaval S. Shah, Anthony M. Rush, Sulayman D. Dib-hajj, Joel A. Black, Lynda Tyrrell, Stephen G Waxman
    Abstract:

    The upregulation of voltage-gated sodium channel Nav1.3 has been linked to hyperexcitability of axotomized dorsal root ganglion (DRG) neurons, which underlies neuropathic pain. However, factors that regulate delivery of Nav1.3 to the cell surface are not known. Contactin/F3, a cell adhesion molecule, has been shown to interact with and enhance surface expression of sodium channels Nav1.2 and Nav1.9. In this study we show that contactin coimmunoprecipitates with Nav1.3 from postnatal day 0 rat brain where this channel is abundant, and from human embryonic kidney (HEK) 293 cells stably transfected with Nav1.3 (HEK-Nav1.3). Purified GST fusion proteins of the N and C termini of Nav1.3 pull down contactin from lysates of transfected HEK 293 cells. Transfection of HEK-Nav1.3 cells with contactin increases the amplitude of the current threefold without changing the biophysical properties of the channel. Enzymatic removal of contactin from the cell surface of cotransfected cells does not reduce the elevated levels of the Nav1.3 current. Finally, we show that, similar to Nav1.3, contactin is upregulated in axotomized DRG neurons and accumulates within the neuroma of transected sciatic nerve. We propose that the upregulation of contactin and its colocalization with Nav1.3 in axotomized DRG neurons may contribute to the hyper-excitablity of the injured neurons.

Gary Matthews - One of the best experts on this subject based on the ideXlab platform.

  • Reduced expression of Nav1.6 sodium channels and compensation by Nav1.2 channels in mice heterozygous for a null mutation in Scn8a
    Neuroscience Letters, 2008
    Co-Authors: Ana V. Vega, Diane Henry, Gary Matthews
    Abstract:

    Abstract The voltage-gated sodium channel α subunit Nav1.6, encoded by the Scn8a gene, accumulates at high density at mature nodes of Ranvier of myelinated axons, replacing the Nav1.2 channels found at nodes earlier in development. To investigate this preferential expression of Nav1.6 at adult nodes, we examined isoform-specific expression of sodium channels in mice heterozygous for a null mutation in Scn8a. Immunoblots from these +/− mice had 50% of the wild-type level of Nav1.6 protein, and their optic-nerve nodes of Ranvier had correspondingly less anti-Nav1.6 immunofluorescence. Protein level and nodal immunofluorescence of the Nav1.2 α subunit increased in Scn8a+/− mice, keeping total sodium channel expression approximately constant despite partial loss of Nav1.6 channels. The results are consistent with a model in which Nav1.6 and Nav1.2 compete for binding partners at sites of high channel density, such as nodes of Ranvier. We suggest that Nav1.6 channels normally occupy most of the molecular machinery responsible for channel clustering because they have higher binding affinity, and not because they are exclusively recognized by mechanisms for transport and insertion of sodium channels in myelinated axons. The reduced amount of Nav1.6 protein in Scn8a+/− mice is apparently insufficient to saturate the nodal binding sites, allowing Nav1.2 channels to compete more successfully.

  • polarized distribution of ion channels within microdomains of the axon initial segment
    The Journal of Comparative Neurology, 2007
    Co-Authors: Audra Van Wart, James S. Trimmer, Gary Matthews
    Abstract:

    Voltage-gated sodium (Nav) channels accumulate at the axon initial segment (IS), where their high density supports spike initiation. Maintenance of this high density of Nav channels involves a macromolecular complex that includes the cytoskeletal linker protein ankyrin-G, the only protein known to bind Nav channels and localize them at the IS. We found previously that Nav1.6 is the predominant Nav channel isoform at IS of adult rodent retinal ganglion cells. However, here we report that Nav1.6 immunostaining is consistently reduced or absent in short regions of the IS proximal to the soma, although both ankyrin-G and pan-Nav antibodies stain this region. We show that this proximal IS subregion is a unique axonal microdomain, containing an accumulation of Nav1.1 channels that are spatially segregated from the Nav1.6 channels of the distal IS. Additionally, we find that axonal Kv1.2 potassium channels are present within the distal IS, but are also excluded from the Nav1.1-enriched proximal IS microdomain. Because ankyrin-G was prominent in both proximal and distal subcompartments of the IS, where it colocalized with either Nav1.1 or Nav1.6, respectively, mechanisms other than association with ankyrin-G must mediate differential targeting of Nav channel subtypes to achieve the spatial precision observed within the IS. This precise arrangement of ion channels within the axon initial segment is likely an important determinant of the firing properties of ganglion cells and other mammalian neurons. J. Comp.

  • Expression of sodium channels Nav1.2 and Nav1.6 during postnatal development of the retina
    Neuroscience Letters, 2006
    Co-Authors: Audra Van Wart, Gary Matthews
    Abstract:

    During the second and third postnatal weeks, there is a developmental switch from sodium channel isoform Nav1.2 to isoform Nav1.6 at initial segments and nodes of Ranvier in rat retinal ganglion cells. We used quantitative, real-time PCR to determine if the developmental appearance of Nav1.6 channels is accompanied by an increase in steady-state level of Nav1.6 mRNA in the retina. Between postnatal day 2 (P2) and P10, Nav1.6 levels did not change, but between P10 and P19, there was an approximately three-fold increase in Nav1.6 transcript levels. This coincides with the appearance of Nav1.6 channels in the retina and optic nerve. The steady-state level of Nav1.2 mRNA also increased during this same period, which suggests that the rise in Nav1.6 may be part of a general increase in sodium channel transcripts at about the time of eye opening at P14. The results are consistent with a developmental increase in steady-state transcripts giving rise to a corresponding increase in sodium channel protein expression.

  • functional specialization of the axon initial segment by isoform specific sodium channel targeting
    The Journal of Neuroscience, 2003
    Co-Authors: Tatiana Boiko, Audra Van Wart, James S. Trimmer, John H Caldwell, Rock S Levinson, Gary Matthews
    Abstract:

    Voltage-dependent sodium channels cluster at high density at axon initial segments, where propagating action potentials are thought to arise, and at nodes of Ranvier. Here, we show that the sodium channel Nav1.6 is precisely localized at initial segments of retinal ganglion cells (RGCs), whereas a different isoform, Nav1.2, is found in the neighboring unmyelinated axon. During development, initial segments first expressed Nav1.2, and Nav1.6 appeared later, approximately in parallel with the onset of repetitive RGC firing. In Shiverer mice, Nav1.6 localization at the initial segment was unaffected, although Nav1.6 expression was severely disrupted in the aberrantly myelinated optic nerve. Targeting or retention of Nav1.6 requires molecular interactions that normally occur only at initial segments and nodes of Ranvier. Expression at nodes but not initial segments exhibits an additional requirement for intact myelination. Because of their high density at the initial segment, Nav1.6 channels may be crucial in determining neuronal firing properties.

Ruth E Westenbroek - One of the best experts on this subject based on the ideXlab platform.

  • localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry
    Journal of Molecular and Cellular Cardiology, 2013
    Co-Authors: Ruth E Westenbroek, Sebastian Bischoff, Ying Fu, Sebastian K G Maier, William A Catterall, Todd Scheuer
    Abstract:

    Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, Nav1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-Nav1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.

  • mice lacking sodium channel β1 subunits display defects in neuronal excitability sodium channel expression and nodal architecture
    The Journal of Neuroscience, 2004
    Co-Authors: Chunling Chen, Ruth E Westenbroek, Yanjun Chen, Xiaorong Xu, Chris A Edwards, Dorothy R Sorenson, Dyke P Mcewen, Heather A Omalley, Vandana Bharucha, Laurence S Meadows
    Abstract:

    Sodium channel β1 subunits modulate α subunit gating and cell surface expression and participate in cell adhesive interactions in vitro . β1(-/-) mice appear ataxic and display spontaneous generalized seizures. In the optic nerve, the fastest components of the compound action potential are slowed and the number of mature nodes of Ranvier is reduced, but Nav1.6, contactin, caspr 1, and Kv1 channels are all localized normally at nodes. At the ultrastructural level, the paranodal septate-like junctions immediately adjacent to the node are missing in a subset of axons, suggesting that β1 may participate in axo-glial communication at the periphery of the nodal gap. Sodium currents in dissociated hippocampal neurons are normal, but Nav1.1 expression is reduced and Nav1.3 expression is increased in a subset of pyramidal neurons in the CA2/CA3 region, suggesting a basis for the epileptic phenotype. Our results show that β1 subunits play important roles in the regulation of sodium channel density and localization, are involved in axo-glial communication at nodes of Ranvier, and are required for normal action potential conduction and control of excitability in vivo .

  • an unexpected role for brain type sodium channels in coupling of cell surface depolarization to contraction in the heart
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Sebastian K G Maier, Ruth E Westenbroek, Todd Scheuer, Kenneth A Schenkman, Eric O Feigl, William A Catterall
    Abstract:

    Voltage-gated sodium channels composed of pore-forming α and auxiliary β subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac pore-forming α subunit isoform Nav1.5 is preferentially localized in intercalated disks, whereas the brain α subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with β scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.

Todd Scheuer - One of the best experts on this subject based on the ideXlab platform.

  • localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry
    Journal of Molecular and Cellular Cardiology, 2013
    Co-Authors: Ruth E Westenbroek, Sebastian Bischoff, Ying Fu, Sebastian K G Maier, William A Catterall, Todd Scheuer
    Abstract:

    Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, Nav1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-Nav1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.

  • Functional Properties and Differential Neuromodulation of Nav1.6 Channels
    Molecular and Cellular Neuroscience, 2008
    Co-Authors: Yanjun Chen, Todd Scheuer, Frank H. Yu, Elizabeth M. Sharp, Daniel Beacham, William A Catterall
    Abstract:

    The voltage-gated sodium channel Nav1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for Nav1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between NaV1.6 and Nav1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Nav1.6 channels, they conducted substantially larger persistent sodium currents than Nav1.2 channels, and they were much less sensitive to inhibition by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Nav1.6 channels in neurons, was not observed for NaV1.6 expressed alone or with the auxiliary β4 subunit. The unique properties of NaV1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.

  • an unexpected role for brain type sodium channels in coupling of cell surface depolarization to contraction in the heart
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Sebastian K G Maier, Ruth E Westenbroek, Todd Scheuer, Kenneth A Schenkman, Eric O Feigl, William A Catterall
    Abstract:

    Voltage-gated sodium channels composed of pore-forming α and auxiliary β subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac pore-forming α subunit isoform Nav1.5 is preferentially localized in intercalated disks, whereas the brain α subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with β scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.

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